Placental proteins are those proteins expressed during pregnancy by the human placenta. The ability to detect the presence and concentrations of these proteins has the potential to provide a reliable diagnostic marker of fertilisation, implantation and pregnancy prognosis.
A number of placental proteins have now been isolated and at least partially characterised. These include--human chorionic gonadotropin (hCG), pregnancy-specific .beta..sub.1 --glycoprotein (SP1), placental protein 5 (PP5), early pregnancy factor (EPF), and pregnancy-associated plasma protein-A (PAPP-A).sup.1.
These proteins are detectable, in maternal blood, at various stages during pregnancy. For example, EPF activity is detectable within 24 hours after conception. HCG is measurable just after implantation, at about 9 to 11 days post-ovulation, SP1 is detectable from 18 to 23 days post-ovulation. In singleton pregnancies, PAPP-A can be detected approximately 28-32 days post-ovulation.sup.2.
Placental proteins are also detectable for varying periods during pregnancy. For example, EPF is detectable at least for the first half of pregnancy, whereafter activity declines until it is totally absent during the third trimester in some women. HCG levels rise rapidly to peak at about 8 to 12 weeks gestation. The levels of SP1 rise exponentially with peak concentrations being reached at term pregnancy. Like SP1, PAPP-A concentrations also rise exponentially in the first trimester of pregnancy to peak at term.sup.2.
Whilst it has been suggested to measure the presence of placental proteins for early detection of pregnancy (for example, see European Application 316919), there is a growing body of documented evidence that at least some placental proteins, particularly PAPP-A, may be used to predict pregnancy viability, including early pregnancy failure, extra-uterine gestations, aneuploid and/or abnormal pregnancies, such as Down's Syndrome.sup.3 and Cornelia de Lange Syndrome.sup.4.
PAPP-A, first described almost two decades ago.sup.1, is a large zinc containing glycoprotein, rich in carbohydrate, with many physicochemical similarities to a 2-macroglobulin.sup.5. It has been detected in maternal circulation.sup.5, pre-ovulatory ovarian follicular fluid.sup.6, in seminal plasma.sup.7 and blood of patients with trophoblastic disease.sup.8.
PAPP-A is a homotetramer, with each monomeric subunit having a molecular weight of approximately 200 kDa. The subunits are linked by disulphide bonds to form dimers of approximately 400 kDa. Native PAPP-A consists of two dimers linked by Van der Waals (ionic) forces. Native PAPP-A has a molecular weight of approximately 820 kDa, regardless of whether it is derived from follicular fluid, seminal plasma, oncological or normal placental tissue.sup.7. The mature protein has a 2-.beta.1 electrophoretic mobility, with an isoelectric point of approximately 4.2-4.5.sup.9. It is a non-competitive and potent inhibitor of human granulocyte elastase.sup.10.
It has been suggested that the biological function of PAPP-A is to act as a local protective barrier against host (maternal) phagocytic-proteolytic defences to either inseminated sperm or the developing feto-placental unit.sup.11. This may be due to PAPP-A forming a protective sheet around the chorionic villus at the utero-placental interface.sup.12. Disruption of this protective layer may explain the correlation between depressed PAPP-A levels and pregnancy failure. PAPP-A may also play a role in zinc homeostasis.sup.5.
Schindler and Bischof.sup.13 suggested that the protein was ubiquitous and, therefore, of little practical use in pregnancy viability diagnosis..sup.14,15 However, it was subsequently shown that these results were due to impure PAPP-A isolates, due to the difficulties in isolating PAPP-A free of a 2-macroglobulin, and polyspecific antisera.sup.16.
Sinosich et al..sup.3 first suggested that a depressed or undetectable PAPP-A level in maternal blood was diagnostic of pregnancy failure. Later, Sinosich et al..sup.17 showed that, of five successful in vitro fertilisation volunteers, three patients with normal pregnancy outcome had circulating PAPP-A levels within the 80% confidence limits of the normal range. By contrast, circulating PAPP-A levels in the patient who spontaneously aborted at seventeen weeks were below the tenth percentile throughout the entire gestation. In the fifth patient, who had a ruptured ectopic pregnancy, PAPP-A could not be detected at any stage during the pregnancy. These findings were complimented by Westergaard et al. (1983).sup.18, who reported that, in a sample group of 51 patients, who conceived spontaneously, with vaginal bleeding in the first half of pregnancy, concentrations of PAPP-A were consistently lower in pregnancies which failed. Similarly, Sinosich et al. (1985).sup.19 showed that, in a group of 21 women who conceived by in vitro fertilisation, PAPP-A levels were consistently depressed, for many weeks, in those women whose pregnancies failed. The same group showed that, of forty seven serum samples obtained from patients with a tubal pregnancy, only two were positive for PAPP-A, indicating that severely depressed or undetectable serum PAPP-A levels were an aid in the diagnosis of extra-uterine pregnancy..sup.20
In 1990, Brambati et al..sup.21 reported that first trimester maternal serum concentrations of PAPP-A were low in pregnancies associated with Down's syndrome. Later, Wald et al. (1992).sup.3 confirmed that PAPP-A concentration was significantly lower in women with Down's syndrome pregnancies compared to PAPP-A levels in a control group of normal pregnancies.
It has also been reported that PAPP-A was detected in the circulation of patients with hydatiform mole.sup.2, suggesting a potential role for PAPP-A quantification in diagnosis and management of certain tumours.
These findings demonstrate the potential diagnostic value of measuring PAPP-A levels for monitoring feto-placental status. Moreover, in view of the increasing use of in vitro fertilisation techniques, and the relatively high proportion of early pregnancy failures associated with these techniques, the measurement of PAPP-A levels to monitor pregnancy viability and thereby minimise patient trauma is clinically advantageous.
Lin. et al. (1974).sup.22 described the use of an electroimmunoassay to measure PAPP-A levels in advanced pregnancy. This assay was insensitive and limited to latter stages of pregnancy. Sinosich et al. (1982).sup.2 and (1984).sup.6 described the first sensitive radioimmunoassay (RIA) which detected PAPP-A in serum obtained from first trimester pregnancies. This assay used radioactively labelled purified PAPP-A together with rabbit anti-human PAPP-A antiserum. The sensitivity of this RIA (2.9 .mu.g/L) enabled PAPP-A detection in maternal blood after the first six weeks of pregnancy. The assay made it possible to detect PAPP-A in other fluids (amniotic fluid, seminal plasma, follicular fluid, gestational trophoblastic disease, culture media), previously beyond the limits of detection. This assay also made it possible to study the kinetics and physiology of maternal PAPP-A levels in the first trimester of pregnancy, a crucial stage for feto-placental development.
The PAPP-A RIA differed from standard protocols in that molecular size of tracer (.sup.125 I-PAPP-A; Mr 820 kDa) and immune complexes required modification of the separation phase. Optimal separation of antibody-bound from antibody-free tracer was achieved with second antibody--7.5%(w/v) polyethylene glycol (PEG) solution, in the ratio of 2:1, 2nd antibody-PEG to assay reaction volume. Under these conditions, assay blank values could be reduced to 5-7%, whilst maximum binding would approach 60-70%.
The development of sensitive and reliable techniques for measuring PAPP-A is dependent on being able to isolate the protein in a sufficiently pure form and/or the generation of monospecific antibodies.
A number of methods for isolating and purifying PAPP-A have been described previously. For example, Lin. et al. (1974).sup.23 describe a procedure based on classical protein fractionation technology. This procedure utilised:
i) solubility; PA1 ii) charge; and PA1 iii) size. PA1 i) solubility; PA1 ii) charge; PA1 iii) lectin affinity; PA1 iv) size fractionation; and PA1 v) negative immunoaffinity. PA1 i) solubility; PA1 ii) positive immunoaffinity chromatography (immobilised anti-PAPP-A antibodies); and PA1 iii) size or charge, respectively. PA1 Glu-Ala-Arg-Gly-Ala-Pro-Glu-Glu-Pro-Ser-Pro-Pro-Ser [SEQ ID NO:9] PA1 (i) applying the sample to a positive affinity chromatography on heparin-Sepharose; PA1 (ii) size fractionating the fractions obtained from step (i) on a gel filtration column; and PA1 (iii) applying the fractions obtained from step (ii) to an anion exchange column; and PA1 (iv) applying the fractions obtained from step (iii) to a matrix with immobilized antibodies. PA1 (i) contacting the sample with a PAPP-A capture phase which consists of one or more of the following: PA1 (ii) contacting the immobilised or captured PAPP-A sample with a monoclonal antibody, of the fifth aspect of the invention, labelled with a detectable marker; PA1 (iii) incubating the sample and the labelled antibody to permit the labelled antibody to bind to any PAPP-A in the sample and; PA1 (iv) detecting the labelled antibody.
This procedure was technically cumbersome and resulted in a low yield of impure material.
Bischof (1979).sup.24 described a method of isolating and purifying PAPP-A. This procedure utilised:
This was even more cumbersome than the earlier procedure. The final product was still impure and yields remained low.
Sutcliffe et al.(1979).sup.25 and Folkersen et al. (1981).sup.26 described a purification method which used;
Step (ii) is detrimental to PAPP-A integrity and neither procedure resulted in pure PAPP-A. Although the final yield was improved by this method, the quality of the protein yield was sacrificed.
Based on an interaction with heparin (Sinosich et al. 1981).sup.27, Sinosich et al. (1982).sup.10 reported the first application of heparin-Sepharose for PAPP-A purification. For the first time it was possible to prepare a high yield (22%) of highly purified PAPP-A. However, by more current and stringent criteria, this preparation was also found to be impure.